Immunoreactive Leukotriene B4 Research Articles

Histamine stimulates alveolar macrophages to release neutrophil and monocyte chemotactic activity

Histamine and serotonin are important inflammatory mediators in the pathophysiology of asthma, and asthmatic patients have higher plasma histamine and serotonin levels than non-asthmatic control subjects. Alveolar macrophages (AMs) synthesize and secrete a large number of substances that play a key role in acute and chronic inflammation including asthma. We postulated that AMs might release chemotactic activity for neutrophils and monocytes in response to histamine or serotonin. To test this hypothesis, bovine AMs were cultured, and the supernatant fluids were evaluated for neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) by a blind well chamber technique. AMs released chemotactic activity in response to histamine and serotonin in a dose- and time-dependent manner (P <.05). Partial characterization and molecular sieve column chromatography revealed that low-molecular-weight lipid-soluble activity was predominant. Lipoxygenase inhibitors significantly blocked the release of chemotactic activity. Leukotriene B4 receptor antagonists blocked the chemotactic activity. Immunoreactive leukotriene B4 significantly increased in supernatant fluids in response to histamine and serotonin. The receptor responsible for the release of chemotactic activity in response to histamine was the H2 receptor. These data demonstrate that AMs release NCA and MCA in response to histamine or serotonin (or both) and may modulate the inflammatory cell recruitment into the lung. (J Lab Clin Med 2001;138:226-35)

The Anti-inflammatory Effect of FR188582, a Highly Selective Inhibitor of Cyclooxygenase-2, With an Ulcerogenic Sparing Effect in Rats

The anti-inflammatory and ulcerogenic effects of FR188582, 3-chloro-5-[4-(methylsulfonyl) phenyl]-1-phenyl-1H-pyrazole, were investigated. In a recombinant human cyclooxygenase (COX) enzyme activity, FR188582 inhibited COX-2 with an IC50 value of 0.017 μM, and the inhibition of prostaglandin (PG) E2 formation by FR188582 was over 6000 times more selective for COX-2 than COX-1. Oral administration of FR188582 dose-dependently inhibited adjuvant arthritis. This effect was threefold more potent than that of indomethacin. FR188582 and indomethacin dose-dependently suppressed the formation of immunoreactive PGE2, but not immunoreactive leukotriene (LT) B4, in arthritic paw. Unlike indomethacin, FR188582 did not induce visible gastric lesions in rats at doses up to 32 mg /kg, p.o. Furthermore, FR188582 did not inhibit the level of immunoreactive PGE2 and immunoreactive 6-keto PGFi1α in rat gastric mucosa. These results suggest that FR188582, a highly selective COX-2 inhibitor, has a potent anti-inflammatory effect mediated by inhibition of PGE2 in inflamed tissues. The safety profile of FR188582 appears to be improved over the safety profile of indomethacin.

Acetylcholine stimulates alveolar macrophages to release inflammatory cell chemotactic activity.

Neurological transmitters including ACh, substance P (SP), and calcitonin gene-related peptide (CGRP) play an important role in regulating airway tone, and increased bronchial reactivity to cholinergic stimulation is a well-recognized phenomenon in patients with bronchial asthma. We postulated that ACh, SP, and CGRP might stimulate alveolar macrophages (AMs) to release neutrophil, monocyte, and eosinophil chemotactic activities. To test this hypothesis, bovine AMs were isolated by bronchoalveolar lavage and cultured. AMs released chemotactic activities in response to ACh in a dose- and time-dependent manner (P < 0.05). However, SP and CGRP did not stimulate bovine AMs. Checkerboard analysis revealed that these released activities were predominantly chemotactic. Partial characterization and molecular-sieve column chromatography revealed that low-molecular-weight lipid-soluble activity was predominant. Lipoxygenase inhibitors significantly blocked the release of chemotactic activities (P < 0.05). Leukotriene B4- and platelet-activating factor-receptor antagonists blocked the chemotactic activities. Immunoreactive leukotriene B4 significantly increased in supernatant fluids in response to ACh (P < 0.05), but platelet-activating factor did not. The receptor responsible for the release of the chemotactic activities was the muscarinic M3 receptor. These data demonstrate that ACh stimulates AMs to release lipoxygenase-derived chemotactic activities and plays a role in inflammatory cell recruitment into the airway.

Pharmacology of the 5-lipoxygenase inhibitors BAY Y 1015 and BAY X 1005 in the horse.

Calcium ionophore A23187 induced time and concentration dependent production of immunoreactive leukotriene (LT) B4 by equine heparinized whole blood in vitro. Time dependent production of immunoreactive LTB4 by equine neutrophils and immunoreactive LTC4 by equine eosinophils in vitro was also demonstrated. The 5-lipoxygenase activating protein (FLAP) inhibitors, BAY X 1005 and BAY Y 1015, produced concentration dependent inhibition of ionophore-induced LTB4 synthesis by equine whole blood (mean +/- SEM IC50s n = 5; 6.14 +/- 0.28 microM vs. 12.30 +/- 0.75 microM for BAY Y 1015 and BAY X 1005, respectively) and neutrophils (mean +/- SEM IC50s n = 5; 0.003 +/- 0.001 microM vs. 0.045 +/- 0.021 microM for BAY Y 1015 and BAY X 1005, respectively) and LTC4 synthesis by equine eosinophils (mean +/- SEM IC50s n = 5; 0.0036 +/- 0.0002 microM and 0.108 +/- 0.023 microM for BAY Y 1015 and BAY X 1005, respectively) in vitro. In all three assays, BAY Y 1015 was more potent than BAY X 1005, and for both compounds much higher concentrations were required to inhibit LT synthesis by whole blood compared to isolated neutrophils and eosinophils. Plasma concentration-time relationships and pharmacokinetic parameters for BAY Y 1015 administered intravenously and orally to six horses at a dosage of 10 mg/kg in a two period cross-over study were established. The study also evaluated the anti-inflammatory properties of BAY Y 1015 and its ability to inhibit ex vivo whole blood LTB4 synthesis and in vivo LTB4 synthesis in a tissue cage model of acute inflammation. At this dosage, BAY Y 1015 failed to significantly inhibit immunoreactive LTB4 synthesis or the oedema produced by intradermal injection of the mild irritant, carrageenan.

Bradykinin stimulates alveolar macrophages to release neutrophil, monocyte, and eosinophil chemotactic activity.

Bronchial asthma is accompanied with inflammatory cell infiltration in the airway. Because kinin activity was detected in bronchoalveolar lavage fluid (BALF) from asthmatic patients, and because the concentrations of kallikrein and kinins in BALF increased after allergen challenge, we evaluated the potential that bradykinin (BK) might stimulate alveolar macrophages (AM) to release neutrophil, monocyte, and eosinophil chemotactic activity (NCA, MCA, and ECA). To test this hypothesis, bovine AM were isolated by bronchoalveolar lavage and cultured. The supernatant fluids of AM were tested for chemotactic activity by a blind well chamber technique. AM released NCA, MCA, and ECA in response to BK in a dose-and time-dependent manner (p < 0.01). The released activities were chemotactic by checkerboard analysis. Partial characterization and molecular sieve column chromatography revealed that these released activities were heterogeneous, suggesting predominant low-m.w. lipid soluble activity and weak high-m.w. peptide activity. Lipoxygenase inhibitors blocked the release of chemotactic activities (p < 0.01). The chemotactic activities were partly inhibited by leukotriene B4 (LTB4) and platelet-activating factor (PAF) receptor antagonists (p < 0.05, respectively). Immunoreactive LTB4 significantly increased in supernatant fluids in response to BK (p < 0.05), but PAF was detected only two out of six samples stimulated by BK. The receptor responsible for the release of LTB4 involved both BKB1 and BKB2 receptors. These data suggest that BK may stimulate AM and play a role in bronchial inflammation by recruiting inflammatory cells into the airway.

Nasal neutrophilia and release of myeloperoxidase induced by nasal challenge with platelet activating factor: Different degrees of responsiveness in atopic and nonatopic subjects

Nasal challenge with platelet activating factor (PAF) is able to induce local neutrophilia, with a different degree of responsiveness in atopic subjects and in nonatopic subjects. We investigated whether nasal accumulation of neutrophils induced by PAF is accompanied by the release of neutrophil-derived mediators. Nasal lavages were performed before and after challenge with PAF (500 nmol), lyso-PAF (500 nmol), and saline solution in 10 patients with allergic rhinitis and 10 normal subjects to evaluate changes in neutrophil counts and the release of myeloperoxidase (MPO) and immunoreactive leukotriene B4. PAF caused neutrophilia, which appeared after 30 minutes in atopic subjects and after 3 hours in nonatopic subjects. Furthermore, when compared with saline insufflation, PAF caused a significant release of MPO in the nasal lavage fluids collected 30 minutes, 3 hours, and 24 hours after challenge in atopic subjects and 3 hours after challenge in nonatopic subjects, with higher values in the former than in the latter. Neutrophil counts correlated with MPO levels in the nasal lavages collected after PAF challenge. A lower degree of neutrophilia was found 3 hours after stimulation with lyso-PAF in both groups of subjects, with a marginal release of MPO in atopic subjects only. No significant increase of immunoreactive leukotriene B4 levels in nasal lavages was found after challenge with either PAF or lyso-PAF. These results indicate that PAF-induced neutrophilia in the nose is accompanied by the release of MPO, which appears earlier and is more marked in atopic subjects than in nonatopic subjects.

Refractory ceramic fibers activate alveolar macrophage eicosanoid and cytokine release

Refractory ceramic fiber has been developed for industrial processes requiring materials with high thermal and mechanical stability. To evaluate the biological activity of this fiber, rat alveolar macrophages were exposed for < or = 24 h to 0-1,000 micrograms/ml of refractory ceramic fiber, crocidolite asbestos, silica (fibrogenic particles), or titanium dioxide (a nonfibrogenic particle), and eicosanoid, tumor necrosis factor-alpha (TNF), and lactate dehydrogenase release were measured. Particle dimensions were determined by electron microscopy. Radioactivity coeluting with leukotriene B4 (LTB4) and immunoreactive LTB4 and TNF release increased after refractory ceramic fiber and were similar in magnitude after asbestos but less than after silica. For example, the total [3H]eicosanoid release increased 3.9-fold after refractory ceramic fiber, 4.6-fold after asbestos, and 8.7-fold after silica. Refractory ceramic fiber and asbestos also have similar particle dimensions (diameter, length, and surface area). Inasmuch as macrophage-derived LTB4 and TNF are potent mediators in inflammatory events, including migration and activation of neutrophils, these findings suggest that refractory ceramic fiber can activate macrophages in vitro to release mediators relevant to in vivo findings of inflammation and fibrotic lung disease in laboratory animals.

Metabolic disposition of leukotriene B4 (LTB4) and oxidation-resistant analogues of LTB4 in conscious rabbits.

1. The kinetics of leukotriene B4 (LTB4), after single i.v. injections of doses of 0.1 to 1 micrograms kg-1, were investigated in conscious rabbits and compared with those of the omega- and beta-oxidation resistant bioactive analogues, 20, 20, 20-trifluoro-LTB4 (20-F3-LTB4) and 3-thio-LTB4, respectively. 2. Immunoreactive LTB4 (IR-LTB4) elimination was first-order, as shown by a constant systemic clearance (ClLTB4) and a proportional increase in the area under the curve (AUC) of the plasma concentration versus time curve over the dose-range studied. Our results showed a good correlation between observed steady-state plasma concentrations (Css) of IR-LTB4 after continuous infusion of LTB4 and those predicted by using the mean estimated ClLTB4 of 93 +/- 4 ml min-1 kg-1, further confirming the linearity of IR-LTB4 elimination. 3. The half-life (t1/2) or IR-LTB4 increased from 0.47 +/- 0.02 to 0.63 +/- 0.04 min as a consequence of a change in the apparent volume of distribution (Vd) from 72 +/- 5 to 109 +/- 13 ml kg-1, for the 0.1 and 1 micrograms kg-1 doses injected, respectively. 4. Single i.v. injections of [3H]-LTB4 (4.7 ng kg-1) were administered, and the decay of plasma [3H]-LTB4 following h.p.l.c. purification was used to estimate the kinetic parameters. The kinetic parameters of [3H]-LTB4 were characterized by a mean systemic clearance (Cl) of 96 +/- 11 ml min-1 kg-1, a t1/2 of 0.53 +/- 0.03 min, and an apparent Vd of 85 +/- 9 ml kg-1, similar to the parameters obtained after LTB4 boluses. 5. The disposition of LTB4 analogues, whether resistant to Omega- or to Beta-oxidation in vitro, did not differ significantly from the disposition of the LTB4 molecule. The half-lives of 20-F3-LTB4 and 3-thio-LTB4 in the circulation were 0.52 +/- 0.07 min and 0.70 +/- 0.11 min, respectively.6. In summary, our results showed that LTB4, as well as Omega-oxidation- and Beta-oxidation-resistant analogues were cleared very rapidly from the rabbit circulation and indicate that in situ, metabolism in blood is not a rate-limiting factor for the elimination of LTB4.

Do infiltrating neutrophils contribute to the pathogenesis of indomethacin induced ulceration of the rat gastric antrum?

The potential involvement of neutrophils in the pathogenesis of indomethacin induced ulceration of the gastric antrum in the re-fed rat was studied. Indomethacin was associated with a time dependent increase in the extent and severity of ulceration, blood neutrophilia, neutrophil infiltration into the gastric antrum, and calcium ionophore induced immunoreactive leukotriene B4 (LTB4) release from the antrum ex vivo. Neutrophil infiltration into the antrum was detectable 1 hour after dosing with indomethacin, at which time damage was apparent microscopically but not macroscopically. Thus, cell infiltration may contribute to the development, if not the initiation, of ulceration. Consistent with this suggestion, oral dexamethasone (5 mg/kg) significantly attenuated indomethacin induced ulceration, the associated neutrophil infiltration, and calcium ionophore induced immunoreactive leukotriene B4 release from the gastric antrum and whole blood ex vivo, although the blood neutrophilia was unaffected. These results suggest that indomethacin induced ulceration of the rat gastric antrum may have a dependence on neutrophil infiltration for its pathogenesis.

Hemoprotein-dependent production of a neutrophil-activating factor from arachidonic acid.

Hemoproteins have been suggested to contribute to various forms of tissue injury by catalyzing the peroxidation of lipids. In this study, the ability of hemoglobin to catalyze the production of a neutrophil-activating factor from arachidonic acid was examined. Incubation of arachidonic acid, hydrogen peroxide, and hemoglobin at 37 degrees C for 30 min resulted in the production of a lipid-extractable substance that was chemotactic for neutrophils in vitro and could stimulate leukocyte adherence in vivo. These actions could be inhibited by two leukotriene B4 (LTB4) receptor antagonists. The peroxidation product cross-reacted significantly with an antibody directed against LTB4, but not with an antibody directed against LTC4. The production of this factor was hemoprotein dependent. Immunoreactive LTB4 and biological activity were produced only when hemoglobin, or another hemoprotein, cytochrome c, was present in the reaction mixture. The amount of the factor produced could be increased in a concentration-dependent manner by increasing the amounts of arachidonic acid or hydrogen peroxide in the reaction mixture. The production of this factor could be inhibited by 5-aminosalicylic acid, catalase, or deferoxamine. Separation of the lipid-extractable products of the peroxidation of arachidonic acid on high-performance liquid chromatography revealed that the immunoreactive (with anti-LTB4) and chemotactic substance had a retention time distinct from that of LTB4 and the hydroxyeicosatetraenoic acids. A lipid-extractable substance with significant cross-reactivity to anti-LTB4 could also be produced if plasma was substituted for arachidonic acid in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased concentration of leukotriene B4 but not thromboxane B2 in intestinal lymph of cats during brief ischemia

Mesenteric ischemia stimulates both A delta- and C-fiber afferents to reflexly activate the cardiovascular system. Leukotriene B4 (LTB4) concentration is increased in intestinal mucosa following prolonged ischemia (3 h) followed by reperfusion. Because LTB4 sensitizes afferent nerve endings in the skin, we determined whether LTB4 is produced during brief mesenteric ischemia and thus would be present to sensitize afferent nerve endings in the abdominal visceral region. Cannulas were placed in the portal vein and in a mesenteric lymphatic vessel distal to the lymph node. Mesenteric lymph and portal venous immunoreactive LTB4 (iLTB4) and immunoreactive thromboxane B2 (iTxB2) concentrations were measured before, during, and after 5-7 min of ischemia induced by occlusion of the descending thoracic aorta in cats. Simultaneously, lymph and plasma lactate concentrations were measured. During arterial occlusion, femoral arterial pressure dropped to less than 30 mmHg, and portal venous and mesenteric lymph lactate concentrations were increased significantly (3.3 +/- 0.6 to 6.3 +/- 1.0 mM and 5.2 +/- 0.9 to 7.2 +/- 1.1 mM, respectively, P less than 0.05). During ischemia, iLTB4 concentration increased in lymph from 261 +/- 70 to 424 +/- 102 pg/0.1 ml (P less than 0.05) but did not increase in portal venous blood (135 +/- 26 vs. 168 +/- 44 pg/0.1 ml, control vs. ischemia). iTxB2 concentration was not increased during ischemia in either portal venous blood or lymph (12 +/- 4 to 24 +/- 9 pg/0.1 ml and 19 +/- 7 to 24 +/- 11 pg/0.1 ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute and chronic suppression of leukotriene B4 synthesis ex vivo in neutrophils from patients with rheumatoid arthritis beginning treatment with methotrexate.

To compare the cumulative effects of oral methotrexate (MTX) therapy (after 6-8 weeks) with the acute effects (24 hours after a dose) on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from patients with active rheumatoid arthritis (RA) who were beginning therapy with MTX. Neutrophils and monocytes were isolated from whole blood from 7 patients with RA, immediately before and 24 hours after their first weekly dose of 7.5 mg of MTX, and again after their dose at 6-8 weeks. Total immunoreactive leukotriene B4 (LTB4) formation in neutrophils activated ex vivo with calcium ionophore A23187 was significantly suppressed (by 33%) before the 6-8-week dose, compared with the level before the first dose (mean +/- SEM 8.29 +/- 1.24 ng/10(6) cells at predose 6-8 weeks versus 12.29 +/- 2.13 ng/10(6) cells at predose 1; P = 0.03). Reductions were also observed after the first dose (27%; P = 0.07) and after the 6-8-week dose (43%; P = 0.05) compared with the respective predose levels. MTX treatment produced significant reductions in the total generation of 5-LO pathway products (5-hydroxyeicosatetraenoic acid + 6-trans-LTB4 + LTB4 + omega-oxidation products of LTB4) by calcium ionophore-activated neutrophils, as quantitated by integrated optical density after resolution on reverse-phase high-performance liquid chromatography. Decreases were observed after the first dose (26%; P = 0.025), immediately before the 6-8-week dose (23%; P = 0.05), and after the 6-8-week dose (47%; P = 0.0033) compared with levels before the first dose, and after the 6-8-week dose compared with the level before it (32%; P = 0.04). The generation of LTB4 by calcium ionophore-activated monocytes was not significantly affected by MTX therapy. The significant decreases in the formation of omega-oxidation products of LTB4 and in the total generation of neutrophil 5-LO pathway products in the absence of a significant change in the release of 3H-arachidonic acid or the generation of platelet-activating factor suggest that the activity of the 5-LO enzyme in neutrophils is inhibited. We conclude that weekly oral MTX therapy in patients with active RA inhibits neutrophil 5-LO pathway product generation in a pattern consistent with inhibition of the activity of the 5-LO enzyme; an effect is observed after the first dose. The inhibition of 5-LO is cell-selective and cumulative, with a superimposed incremental inhibition observed after the weekly MTX dose.

Identification of guinea pig eosinophil chemotactic factor of anaphylaxis as leukotriene B4 and 8(S),15(S)-dihydroxy-5,9,11,13(Z,E,Z,E)-eicosatetraenoic acid.

We have purified and characterized the guinea pig eosinophil chemotactic factor of anaphylaxis (ECF-A), an activity previously described in diffusates from sensitized lung challenged with specific Ag that appeared to selectively attract eosinophils from mixed leukocyte populations. Time course studies showed that the release of ECF-A from challenged presensitized guinea pig lung fragments closely paralleled the release of immunoreactive leukotriene B4 (iLTB4) and histamine. However, the majority of ECF-A (greater than 80%) and iLTB4 (greater than 79%) was extractable with the lipid fraction from the methanol wash of Sep-Pak-extracted diffusate, whereas histamine remained in the aqueous phase. A comparable neutrophil chemotactic activity was also found in the methanol extracts of the anaphylactic diffusates. By using a combination of HPLC and specific RIA, greater than 60% of ECF-A was attributable to LTB4. A second eosinophil chemotactic activity was also identified and coeluted (on both reverse phase and straight phase HPLC) with the synthetic standard 8(S),15(S)-dihydroxy-5,9,11,13(Z,E,Z,E)eicosatetraenoic acid (8(S),15(S)-diHETE). This was confirmed as 8(S),15(S)-diHETE by gas chromatography-mass spectrometry. Platelet-activating factor and histamine had negligible activity for guinea pig eosinophils, compared with synthetic LTB4 (p less than 0.05, 10(-9) and 10(-8) M; p less than 0.01, 10(-7) to 5 x 10(-6) M). In addition, synthetic 8(S),15(S)-diHETE had 3 times less activity than LTB4 at optimal chemotactic concentrations (10(-6) and 10(-7) M, respectively). Thus, guinea pig ECF-A appears to be largely attributable to lipoxygenase products of arachidonic acid, namely LTB4 and 8(S),15(S)-diHETE. Because guinea pig ECF-A was equally active on neutrophils (greater than 96% purity), it can no longer be considered a selective eosinophil chemoattractant.

Increased levels of immunoreactive leukotriene B4 in blister fluids of bullous pemphigoid patients and effects of a selective 5-lipoxygenase inhibitor on experimental skin lesions

The immunoreactive leukotriene B4 (i-LTB4) and i-LTC4 content in the blister fluids of patients with bullous pemphigoid (BP) was determined by radioimmunoassay. Their amounts significantly exceeded those noted in superficial dermal burn patients and those in the fluids of suction blisters produced on normal human skin. When either BP blister fluids or BP-IgG from the patient sera were injected into guinea pig skin, neutrophil and eosinophil infiltrates were produced in the dermis. In addition, the dermis was noted to undergo marked edematous change. A single oral administration of a selective inhibitor of 5-lipoxygenase 1 hr before the intracutaneous injection of BP-IgG was found to significantly inhibit cell infiltrates. Furthermore, the inhibitor partly suppressed dermal epidermal separation. On the basis of these results. LTB4 and LTC4 appear to be generated in the skin lesions of BP, the former attracting granulocytes to the dermis and the latter, causing exudation.

Generation of Lipoxygenase Metabolites of Arachidonic Acid by Monolayer Cultures of Tracheal Epithelial Cells and Intact Tracheal Segments from Rabbits

We compared the profile of lipoxygenase metabolites of arachidonic acid (AA) generated by cultured rabbit tracheal epithelial (TE) cells with that produced by intact rabbit tracheal segments at baseline and following addition of exogenous AA or calcium ionophore A23187. Lipoxygenase metabolites in effluent media were resolved by high-pressure liquid chromatography and quantitated by radioimmunoassay for monohydroxyeicosanoid (HETE) and leukotriene (LT) metabolites [5-, 12-, and 15-HETE; LTB4, LTC4, LTD4]. Following incubation with exogenous AA (10 micrograms/ml), cultured TE cells generated immunoreactive products that coeluted with authentic 5-, 12-, and 15-HETE standards. 12-HETE was the predominant metabolite. Whereas the generation of HETEs by TE monolayers was dependent on addition of exogenous AA, intact tracheal segments demonstrated a baseline production of 12-HETE and lesser amounts of 5- and 15-HETE as well as unidentified metabolites with UV absorbance at 280 nm. Incubation of tracheal segments with AA resulted in augmented metabolite production. In cultured TE cells, small quantities of HETEs were present intracellularly esterified to membrane phospholipids or free in the cytosol, and significant increases in free cytosolic 12- and 15-HETE were detected postincubation with AA. Calcium ionophore (5 microM) did not induce significant increases in HETE production in either cultured TE cells or tracheal segments. Minimal or no immunoreactive LTs B4, C4, and D4 were produced by TE monolayers or tracheal segments at baseline or following addition of AA or ionophore. Production of HETEs by cultured TE cells was not associated with decreased viability, release of intracellular lactic dehydrogenase, or loss of cells from the monolayers. Preincubation of monolayer cultures or tracheal segments with 5,8,11,14-eicosatetraynoic acid prior to addition of exogenous AA inhibition metabolite production. Our observations provide further documentation for the generation of lipoxygenase metabolites by TE cells and suggest that the array of metabolites generated by cultured TE cells may not be representative of the entire spectrum of AA metabolites produced by intact native epithelium.

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production.

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)

Low-molecular-weight leukocyte chemotactic factors in psoriatic scales: contribution of lipid-soluble factors to the chemotactic activity.

AbstractPsoriatic scales contain polymorphonuclear leukocyte (PMN) chemotactic factors which presumably are responsible for transepidermal PMN chemotaxis in psoriatic lesions. On molecular sieve chromatography, psoriatic scale extracts revealed PMN chemotactic activity in the peptide fractions eluted near 12 kDa in addition to low molecular weight (MW) chemotactic fractions. PMN chemotactic peptides with MW around 12 kDa that contain a C5‐cleavage product have been characterized as a unique major chemotactic factor in the aqueous components. On the other hand, it is uncertain whether or not lipid‐soluble low MW chemotactic factors, i.e., leukotriene B4 (LTB4) and its related compound 12‐hydroxy‐5,8,10,14‐eicosatetraenoic acid (12‐HETE), play major roles as PMN chemoattractants. In the present study, we found that the chemotactic activity in the aqueous psoriatic extracts was much higher than that in the organic extracts. Chemotactic activity in the low MW chemotactic fractions of psoriatic scale extracts, about half of which was recovered after extraction with ether at acidic pH, was heat‐stable (100°C) and withstood protease digestion, but was partially inactivated by treatment with trypsin. Immunoreactive LTB4 was demonstrated in these fractions. The previous findings and the present results suggest that the lipid‐soluble chemotactic factors, LTB4 and 12‐HETE, which constitute the low MW fraction of the psoriatic scale extracts, do not play as major a role as a PMN chemoattractants as do the chemotactic peptide fractions.

Generation of Leukotriene B4by Human Lung Fragments and Purified Human Lung Mast Cells

Leukotriene B4 (LTB4) is a potent stimulus for neutrophil chemotaxis, aggregation, and activation. Although this mediator has been postulated as a possible stimulus for the neutrophil accumulation seen after mast cell triggering in vivo, the ability of human mast cells to produce this leukotriene has never been described. The purpose of this study was to investigate the ability of purified human lung mast cells and mast cells in lung fragments to generate leukotriene B4 after immunologic (anti-IgE) and nonimmunologic (calcium ionophore, A23187) activation. Release of LTB4 was quantitated by 2 specific radioimmunoassays with biochemical characterization by high performance liquid chromatography. In a first series of experiments using radioimmunoassay 1, purified human lung mast cells (n = 10) released LTB4 in response to both an immunologic and nonimmunologic stimulus in a time- and dose-dependent manner. In response to an optimum concentration of anti-IgE (3 micrograms/ml), mast cells of 4.5 to 98% purity, generated 6.2 +/- 1.7 ng immunoreactive LTB4 (iLTB4)/10(6) mast cells. HPLC characterization revealed that 30 +/- 0.3% of the iLTB4 coeluted with standard synthetic LTB4 in these studies. In a second series of experiments using radioimmunoassay 2, mast cell activation resulted in the release of 0.5 to 1 ng iLTB4/10(6) mast cells with greater than 75% coeluting with synthetic LTB4. Thus, we estimate that human lung mast cells can generate approximately 1 to 2 ng of LTB4 per million mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of immunoreactive leukotriene B4 in uterine lavage fluid from mares with experimentally induced and naturally occurring endometritis.

Acute endometritis was induced in ovariectomized pony mares by infusion of a 1% solution of oyster glycogen. Maximum concentrations of immunoreactive leukotriene B4 in uterine washings coincided with the greatest rate of infiltration of neutrophils into the uterine lumen. Concentrations of immunoreactive leukotriene B4 decreased to basal levels 6 h after infusion and were unaffected by administration of ovarian steroids to ovariectomized mares. Uterine washings from mares with persistent endometritis did not contain significantly different concentrations of leukotriene B4 from genitally normal mares.

Synthesis of arachidonate oxidation products by synovial joint tissues during the development of chronic erosive arthritis

An animal model, antigen-induced arthritis in the rabbit, was used to investigate the synthesis of arachidonic acid oxidation products (eicosanoids) by tissues of the knee joints during the initiation and early phase of this developing chronic destructive lesion. High concentrations of immunoreactive prostaglandin E2 and immunoreactive leukotriene B4 were found to be present in the synovial fluid in the early lesion. These levels rapidly declined, as did the ability of the infiltrating cells of the arthritic joint fluids to synthesize leukotriene B4. Articular cartilage and synovial lining were maintained for 24 hours in nonproliferative organ culture, and the synthesis of eicosanoids was measured by assay of the culture fluids. The synovial lining was the major source of eicosanoids. Synthesis of prostaglandin E2, prostacyclin, thromboxane A2, and leukotriene B4 by the arthritic synovial lining was low during the first few days, but reached maximal values between day 5 and day 15 of disease duration. Maximal eicosanoid production occurred around the time that damage to articular cartilage and bone has been reported to occur, though it is not certain that these two events are linked. It was demonstrated that, in chronic lesions, the arthritic synovial lining was still producing elevated levels of eicosanoids compared with levels in the control tissue.